Connexin43 represents an important regulator for Sertoli cell morphology, Sertoli cell nuclear ultrastructure, and Sertoli cell maturation

The Sertoli cell (SC)-specific knockout (KO) of connexin43 (Cx43) was shown to be an effector of multiple histological changes in tubular morphology, resulting in germ cell loss through to a Sertoli-cell-only (SCO) phenotype and vacuolated seminiferous tubules containing SC-clusters. Our present study focused on the effects of Cx43 loss on SC ultrastructure. Using serial block-face scanning electron microscopy (SBF-SEM), we could confirm previous results. Ultrastructural analysis of Sertoli cell nuclei (SCN) revealed that these appear in clusters with a phenotype resembling immature/proliferating SCs in KO mice. Surprisingly, SCs of fertile wild type (WT) mice contained SCN with a predominantly smooth surface instead of deep indentations of the nuclear envelope, suggesting that these indentations do not correlate with germ cell support or spermatogenesis. SBF-SEM facilitated the precise examination of clustered SCs. Even if the exact maturation state of mutant SCs remained unclear, our study could detect indications of cellular senescence as well as immaturity, emphasising that Cx43 affects SC maturation. Moreover, Sudan III staining and transmission electron microscopy (TEM) demonstrated an altered lipid metabolism in SCs of Cx43 deficient mice.

www.nature.com/scientificreports/ Cx43 plays a key role in the regulation of SC proliferation and maturation [18][19][20] . Moreover, structural alterations of seminiferous tubules occur in the seminiferous epithelium of homozygous adult mutant mice. Tubules display germ cell loss, resulting in the arrest of spermatogenesis at the level of spermatogonia or Sertoli-cell-only (SCO) syndrome. These seminiferous tubules are smaller in diameter compared to those of their WT littermates and are often vacuolated, with intratubular located, clustered SCs 18,[20][21][22] . Furthermore, it was found that SC numbers per seminiferous tubule are increased in SCCx43KO −/− mice 18,21 . Although the tubular structure is altered, it was verified that the integrity of the blood-testis-barrier persists in mutants [23][24][25] . Rat SCs have been reconstructed in three-dimensional level for the first time by Wong and colleagues using time consuming semi-serial thin sectioning techniques and serial reconstruction techniques 26 . Serial block-face scanning electron microscopy (SBF-SEM) is a relatively new technique to process resin-embedded tissue samples. The method offers various advantages compared to conventional transmission electron microscopy (TEM): the slicing and imaging process runs completely automatic, which makes it time-saving for the examiner 27 and creates homogenous images. Together with a large field of view this allows imaging of specimen blocks of a greater volume than with TEM techniques 28 . Since the slice thickness is only a few nanometres, the Z-axis of tissues can be imaged without almost any loss of information, thus specimens could be examined with greater ease not only two-dimensionally, but also in three-dimensional level.

Results
Sudan III staining. In testes of wild type (WT) mice, intracytoplasmic Sudan III-stained granules occurred particularly in interstitial Leydig cells (LC) and were rarely found in SCs (Fig. 1a,d). A similar interstitial distribution pattern was also seen in testes of SCCx43KO −/− mice, but granules were distinctly larger and more abundant. While SCs of WT mice showed only faint Sudan III staining, intratubular SC-clusters in mutants contained SCs with numerous lipid droplets (Fig. 1b,e). Intratubular vacuoles did not contain Sudan III-stained material. SBF-SEM could confirm the distribution pattern of lipid droplets in intratubular cell-clusters of SCCx43KO −/− mice (Fig. 1c,f, Supplementary Video S1).
Counting of lipid droplets in tubule cross-sections by light microscopy resulted in a 5.764-fold higher average number in the 68 days old mutant compared to his WT littermate. This difference was even stronger in the 138 days old SCCx43KO −/− male, who showed a 12.59-fold increase. The evaluation of seminiferous tubules using ImageJ could confirm these results: at the age of 68 days, the number of lipid droplets per tubule cross-section was 5.698-fold higher, at the age of 138 days the increase was 8.675-fold. In addition, using TEM it could be shown that the average number of lipid droplets in SCs at the basal compartment of the seminiferous tubule in mutant Figure 1. Sudan III staining of murine wild type (WT) (a,d) and SCCx43KO −/− (b,e) testes. In WT testes Sudan III-stained granules particularly occur in the cytoplasm of interstitial Leydig cells (LC) (transparent arrows) and rarely in Sertoli cells (SC). A similar distribution pattern is seen in LCs of SCCx43KO −/− mice (transparent arrows), but granules are larger and more numerous. Especially SCs of intratubular SC-clusters also contain abundant Sudan III-stained granules (black arrows). Serial block-face scanning electron microscopy sections of a SCCx43KO −/− testis (c,f). Lipid droplets are concentrated in the tubular SC-clusters (c, white circle). Higher magnification displays that lipids are accumulated in the SC's cytoplasm (f, white arrows). Scale bars (a,c) = 50 µm, (b,d) = 20 µm, (e,f) = 10 µm. www.nature.com/scientificreports/ mice is 3.294-fold higher per field of view. Moreover, having a closer look on lipid droplets using TEM revealed that these lipid droplets are often arranged in small groups in SCs of mutants ( Supplementary Fig. S1c,e,f). These groups did also rarely occur in SCs of WT mice, but seemed to contain less droplets ( Supplementary Fig. S1b). . In addition, SCN of WT mice showed a typical mature phenotype, but, surprisingly, less indentations of the nuclear envelope as expected from literature. SCN mainly had a round to oval shape (Fig. 4a,c,e) and were located near the tubular basement membrane (Fig. 3a). They contained mostly two satellite chromocenters, less common ones, forming the characteristic tripartite nucleolus. No heterochromatin patches could be found near the nuclear membrane. In contrast, seminiferous tubules of SCCx43KO −/− mice contained two types of SCN: basally located SCN with a mature phenotype and SCN of SC-clusters (Fig. 3b, Supplementary Video S1) showing signs of immaturity. SCN of mutants were numerously and deeply intended (Fig. 4b,d,f, Supplementary    www.nature.com/scientificreports/ Video S3). In basally located SCN, nuclear clefts could be found particularly at the basal and apical surface, whereas in clustered SCN, clefts were diffusely distributed over the nuclear surface. In SCCx43KO −/− mice, the most obvious alterations occurred in SCN of intratubular SC-clusters and could be seen as features of immaturity: these SCN had a smaller diameter and a polygonal shape, plus heterochromatin patches along the nuclear envelope ( Fig. 3, insert, Supplementary Video S1).

Special nuclear morphology.
In addition, in one of the SCCx43KO −/− mice, two basally located SCN showing a special phenotype, different from the other SCN, were detected. The first one appeared like two SCN, which were connected via a small nucleoplasmic bridge, forming one distinctly larger SCN, than the others of the investigated tubular section ( Fig. 5a-d). Each of these connected SCN owned a nucleolus, one of which was smaller and associated with the smaller nucleus. While the smaller SCN contained only one satellite chromocenter, the larger one contained six of these. In total, the SCN showed multiple and deep indentations of the nuclear envelope, particularly at the lateral and basal surface. Taken together, the described morphological features indicated that these two SCN were probably nuclei of proliferating SCs. The second SCN showed mostly shallow indentations of the nuclear envelope ( Fig. 5e,g,h). One deep indentation could be found in the SCN's centre (Fig. 5f). Several small heterochromatin patches were distributed along the nuclear envelope. Most obvious are the convoluted, euchromatic nucleolus-like structures at different locations within the SCN.
In order to determine the proliferation status and thus maturation state of SCs in adult mutants more precisely, double-immunofluorescence staining for Sox9 and BrdU was performed ( Fig. 6a-i). Single clustered SCs were immunopositive for both Sox9 and BrdU (Fig. 6d, white arrows), indicating that these cells are proliferating SCs. This phenomenon could so far only be observed in clustered SCs, whereas basally located SCs stained immunonegative for BrdU. However, not every SC-cluster contained BrdU immunopositive SCs (data not shown).

Discussion
Our results highlight that Cx43 plays a crucial role not only for tubular morphology, but also for SC ultrastructure and maturation. Germ cells are embedded in a stage-specific manner between the long cytoplasmic branches of SCs, which extend to the tubular lumen 2,26 . In the present study, seminiferous tubules of SCCx43KO −/− mice were vacuolated and the tubular lumen was filled with entangled SC processes. Since there was no evidence of lipids in tubular vacuoles, the vacuolated phenotype occurs from entangled SC processes, occluding the tubular lumen. The question arises if the convoluted apical SC surface is a result of germ cell absence or a direct effect of SC-specific Cx43 loss. The absence of structural support of differentiated germ cells in SCCx43KO −/− mice In the top row, the nuclear envelope is numerously and deeply indented, particularly at the lateral and basal surface (a-d). Two nucleoli (a-d, black arrows) and several satellite chromocenters (a-d, transparent arrows) can be found. It seems that two SCN are connected via a small bridge (a,b,d), possibly representing a SCN in the late telophase of mitosis. In the bottom row, the SCN shows several heterochromatin patches along the nuclear envelope (e-h, white arrows). One deep indentation of the nuclear envelope is visible in the SCN 's centre (f). The other clefts are more shallow (f,g). Nucleolus-like structures appear at different locations within the SCN (e-h, white asterisks). Scale bars = 5 µm. www.nature.com/scientificreports/ might cause these long disorganized cytoplasmic processes. The fact that various other studies, using different methods and mouse models, resulting likewise in a SC-only phenotype, display a comparable tubular morphology, strengthens the latter hypothesis [29][30][31][32][33] . Following experimental cryptorchism, seminiferous tubules of rats developed vacuoles between neighbouring SCs at the basal compartment of seminiferous tubules. With persistent cryptorchid state, these vacuoles were no longer visible but parallel arranged smooth membrane stacks appeared, which were assumed to be a result of vacuole disintegration 34 . We found similar vacuoles and membrane-like stacks in SCCx43KO −/− mice, which are presumably a consequence of permanent germ cell absence in the adluminal compartment of the seminiferous epithelium. Since the almost complete lack of apical germ cell-SCinteractions in mutants, SCs showed a decrease in height and seemed to have collapsed. SCs are considered to be polarized cells, with a nucleus residing near the basement membrane, except at the time of spermiation 3 . Seminiferous tubules of SCCx43KO −/− mice contained intratubular SC-clusters. SCN of these clusters were distinctly located away from the tubular wall, indicating that these cells were either removed from the basal compartment of the seminiferous epithelium as Sridharan et al. suspected 18 or merely lost their cellular polarity. Since the basement membrane contact is thought to be essential for SC survival 35 , it is more likely that clustered SCs lost their polar phenotype. Senescence appears to have influence on SC polarity, since SCN in ageing rats lose their closeness to the basement membrane without inducing cellular death 36 . Even the fact that cluster size increases with age 20 , supports the theory that SC-clusters might be composed of aged nonpolarized SCs. Some of these Sox9-immunopositive SCs also stained positive for BrdU, indicating proliferation as a sign for immaturity or at least an intermediate differentiation state. However, the remaining question is, what causes SC polarity loss. Previous investigations using XX-sex-reversed mice, showing similar SC-clusters also www.nature.com/scientificreports/ with morphological features of immaturity, hold the absence of germ cells accountable for clustered SCs 33 . Since differentiated germ cells are also absent in SCCx43KO −/− mice, they could be a missing landmark for SC polarity. Moreover, changes in lipid metabolism are considered as another age-related process 37 . In the present study, only few Sudan III-stained granules occurred in SCs of WT mice. Surprisingly, an accumulation of lipid droplets was found in intratubular SC-clusters of same-aged SCCx43KO −/− littermates. Degenerating spermatogenic cells and residual bodies are degraded and digested by SCs through phagocytosis 3,38,39 , forming lipid inclusions. The number and size of lipid droplets in SCs were shown to be stage-dependent, increasing in number during the second half of the seminiferous cycle [39][40][41] . These lipids are predominantly degraded via the β-oxidation pathway, providing an important source for SC-ATP synthesis 42 . Since germ cell numbers are already reduced in neonatal SCCx43KO −/− mice plus few differentiating spermatogonia and early spermatocytes are solely seen in pubertal mice 22 , it is unlikely that lipid droplets occur in SCs of adult mutants due to directly preceding apoptosis of differentiated germ cells. Moreover, no enhanced apoptosis was apparent. Therefore, the increased number of lipid inclusions in SCs of SCCx43KO −/− mice might have another cause. Two possible sources of augmented lipids in clustered SCs are conceivable: (1) an enhanced energy need resulting in an increased accumulation of intracellular lipids as a substrate for ATP synthesis, or (2) a decreased lipid degradation. Since germ cells are mostly absent or seminiferous tubules of mutants display a SCO phenotype, there is a lack of phagocytozeable cells. The question arises, where these lipids come from. Nevertheless, since clustered SCs show signs of enduring proliferation, the former hypothesis of enhanced energy need seems likely. Alternatively, Cx43-loss might directly affect the lipid metabolism in SCs, but further studies are needed to investigate the potentially altered pathways. Nistal et al., who examined the SC morphology in human testes showing SCO syndrome, also found abundant lipid droplets in SCs with deeply infolded nuclei and suggested accelerated SC ageing as a cause for this phenotype 43 . Nevertheless, since clustered SCs of mutants show signs of enduring proliferation, which is a feature of immaturity, it is unlikely that these cells are aged. Besides seminiferous tubules, the testicular interstitium also demonstrated alterations. Lipid droplets were more abundant in the interstitium of mutants compared to WT littermates. Since Noelke et al. confirmed a LC hyperplasia in SCCx43KO −/− mice 44 , increased numbers of interstitial lipid droplets could be considered as confirmation that SC-specific Cx43 loss also has an impact on LCs. The quantification of lipid droplets in SCs resulted in a notably higher number of lipid droplets in mutant mice. This difference was even more prominent in the older mutant. Since it is known that the number of clustered SCs increases with age 18,20 , this could be a possible reason for these findings. To exclude SC-clusters as the sole cause for higher numbers of lipid droplets in SCCx43KO −/− mice, the number of lipid droplets in SCs at the basal compartment of the seminiferous tubule was determined per field of view in TEM pictures. Results show that, indeed, clustered SCs seem to contribute to a large proportion to the elevated number of lipid droplets in Cx43-deficient SCs but the number of droplets is also higher in basally located SCs.
So far, SCs are still considered as postmitotic cells, ceasing their proliferation during puberty [14][15][16][17] , but some researchers queried this opinion 45,46 . Using the present KO mouse model, it was determined that some SCs might be still proliferating in adult SCCx43KO −/− mice 18,20 . From this observation arose the hypothesis that SCs are not terminally differentiated, postmitotic cells, but cells, in which proliferation is only suppressed and can be resumed under specific conditions. In our model, we proved that testes of mutants contain more SCs in comparison to WT mice 18 . Moreover, we found clustered Sox9 immunopositive SCs, which were also stained immunopositive for BrdU, and two basally located SCN with a special and interesting phenotype in one of the investigated SCCx43KO −/− mice. One SCN contained several satellite chromocenters, the other one resembled a nucleus in the late telophase, both representing most likely nuclei of proliferating SCs. Since the appearance of several nucleoli is a feature of immature SCs 7,47 , it could be shown for the first time that seminiferous tubules of adult SCCx43KO −/− mice do not only contain SCs with a mature or intermediate phenotype, but also immature SCs with proliferative characteristics. Even if it was found so far that basal SCs in adult mutants are immunonegative for proliferation markers and markers of immaturity 20 , it could be interesting, if these cells do express these markers anyway, since SBF-SEM revealed SCN with a proliferative or even immature phenotype. Adult SCs from the terminal segments of the seminiferous tubule are shown to be intensively proliferating and building colonies in-vitro. These cells are comparable with clustered SCN of our KO mouse model, possessing atypical nuclei with several heterochromatin patches instead of two distinct satellite chromocenters 48 .
SCs in SC-clusters demonstrate features of both aged mature and immature SCs. The fact that Cx43 is a negative regulator of SC proliferation 49,50 , emphasizes that the absent negative feedback of Cx43 might be responsible for enduring SC proliferation in SCCx43KO −/− mice. By means of double-immunofluorescence for Sox9 and BrdU, we could emphasise the hypothesis of proliferating SCs in adult mutants only for clustered SCs so far, even if some basally located SCs also showed signs of immaturity and features of mitotic cells at ultrastructural level.
Unexpectedly, SCN of WT mice mainly had a round to oval shape and indentations of the nuclear envelope were rarely seen compared to SCCx43KO −/− littermates. Different studies have shown that nuclear infoldings of SCs are regulated by hormones 51,52 . Nuclear clefts do also appear in other cell types and are associated with both physiological and pathological processes. For example, they occur in the dorsal root ganglion as a reaction to trauma and are postulated to be related to an enhanced nuclear metabolism 53 . Moreover, under normal conditions neurons of the neostriatum and pyramidal cells also show indented nuclei 54,55 . In the human and mammalian ovary, infoldings appear physiologically in the nuclei of granulosa cells [56][57][58] . Based on the theory that nuclear indentations are a sign of enhanced cellular activity and some proteins are shown to be higher concentrated within indentations 59 , SCs of mutants might be more active compared to WT mice. Nevertheless, little is known about the indentation's functions. In different cell lines, nuclear invaginations formed channels, which were linked with the nucleolus. It was hypothesized that they work as a nucleolocytoplasmic transfer device 60 or reduce the distance between the nucleolus and the nuclear envelope 61 . As germ cells are mostly absent in SCCx43KO −/− mice, the question arises, what triggers cellular activity of SCs. Based on the fact that single clustered SCs are still proliferating in adult mutants, enduring proliferation is probably a target of enhanced cellular activity as well as www.nature.com/scientificreports/ augmented nucleocytoplasmic transport. The fact that heterochromatin patches are regularly detectable in SCN of SCCx43KO −/− mice and that this phenotype was shown to be coherent with mitotic activity 48 , supports this hypothesis. Moreover, it was shown that nuclear envelope proteins are involved in regulation of heterochromatin formation in mammalian cells 62,63 . Since in the present study, SCN of mutants are significantly more indented than SCN of WT littermates and heterochromatin was arranged alongside the nuclear envelope, it would be interesting to find out if both alterations relate or even though they are mutually dependent.

Conclusion
In summary, the SC-specific deletion of Cx43 leads to both structural and particularly ultrastructural alterations of tubular and SC morphology as well as metabolic changes. Moreover, the hypothesis that single SCs are still proliferating in adult SCCx43KO −/− mice was underlined. The new and exciting technical approach by SBF-SEM provided the opportunity to examine the ultrastructure of a large specimen volume and to reconstruct threedimensional nuclear morphology. Further studies are needed, to investigate potential mechanisms of metabolic alterations and functional backgrounds of morphological changes in SCs of SCCx43KO −/− mice.

Materials and methods
Generation of SCCx43KO −/− mice. Transgenic KO mice lacking Cx43 solely in SCs were generated using the Cre/loxP recombination system. All details of the breeding strategy, PCR genotyping, and confirmation of the Gja1 loss by β-galactosidase immunohistochemistry are described elsewhere 21   Image segmentation and analysis. Processing, reconstruction and analysis was performed using the freeware Microscopy Image Browser (MIB) 64 . Images from SBF-SEM were resampled (2000 × 2000 pixel, 75 nm pixel size) and converted from 16 to 8 bit format to create a smaller dataset, which facilitates further handling on the one hand and still provides sufficient resolution to analyze the structures of interest on the other hand. www.nature.com/scientificreports/ The contrast was adjusted to compensate for variations between the slices. For analysis, image stacks containing 500 images were aligned. Assessment criteria were nuclear shape and size, number of satellite chromocenters, heterochromatin distribution plus SC-localization within the seminiferous tubule.